Project Summary: Double stranded breaks (DSBs) in DNA can be misrepaired, causing gross chromosomal rearrangements (GCRs) such as translocations, large deletions, and chromosome loss. These GCRs are thought to be causative in speciation and human disorders, especially cancer. The causes of DSBs and associated GCRs in most human pathologies have eluded researchers. One potential source of these deleterious DSBs and GCRs may be hybrids that form between RNA and homologous chromosomal DNA sequences. My laboratory has provided important new insights into hybrid formation and hybrid-mediated GCRs using budding yeast as a model system. We developed important new tools: 1) a yeast artificial chromosome (YAC) as a powerful genetic and molecular reporter of hybrid-mediated GCR; 2) a simple cytological assay to detect hybrids in individual cells; and 3) a model locus in the YAC at which we can induce hybrid formation and hybrid-mediated GCRs. Using our model locus, we showed that aberrant RNAs were much more proficient at causing hybrid mediated GCR than canonical mRNAs. In addition, hybrids can form in trans; that is, at a locus distinct from the site of transcript synthesis. Remarkably, the formation of these hybrids is mediated by the RAD51 repair pathway. We propose to exploit these findings to define the parameters in RNA and DNA (sequence, length,etc) that both promote and repress hybrid formation, DSB formation and hybrid-mediated GCR. We also showed that hybrid and GCR formation are greatly elevated in many mutants defective in diverse aspects of RNA biogenesis. We propose to generate the first genomic maps of hybrids associated with GCR in wild type and mutants. These maps, along with the model locus studies, will provide critical insights into how cis and trans factors influene the interplay between transcription, GCRs and genome instability. We identified two anti-hybrid systems. The first utilizes RNase H, an enzyme that degrades RNA in hybrids, and the second uses Srs2, an antagonist of Rad51. These two anti-mutagenic systems have escaped study despite 50 years of intensive research on DNA repair. We propose to elucidate the molecular function and biological impact of these hybrid-removal systems through genetic, biochemical and genomic methods. Finally, the least understood component of hybrid-mediated GCRs is the conversion of hybrids to DSBs. We will use our model locus to develop methods to detect hybrid-mediated DSBs and elucidate their mechanism of formation. The feasibility of the proposed studies is extremely high because of our novel assays for hybrid and GCR analysis and existing powerful approaches available in budding yeast. There is a remarkable conservation between budding yeast and human cells in the processes of RNA and DNA metabolism. Emerging studies are beginning to reveal a similar conservation for hybrid-mediated GCRs. Given these precedents, the experiments in this proposal are likely to be applicable to higher organisms, with the potential to provide important insights into human health, such as cancer and birth defects.